MicroRNAs (miRNAs) are -21 nucleotide noncoding RNAs that posttranscriptionally regulate gene expression in plants and animals. Although miRNA-mediated regulation is clearly essential for proper morphogenesis and differentiation in plants and animals, relatively little is known about the mechanistic basis of miRNA functions during development. Arabidopsis thaliana embryos are an ideal system for characterizing the developmental roles of miRNAs for several reasons, including: 1) miRNAs have important functions during Arabidopsis embryogenesis, 2) the vast majority of Arabidopsis miRNA targets have been confidently predicted, 3) molecular genetic approaches can be efficiently used to characterize miRNA/target interactions in vivo, and 4) there are several advantages to studying pattern formation in Arabidopsis embryos. The proposed research plan therefore utilizes Arabidopsis embryos to characterize the roles of miRNAs in fundamental cell fate decisions with the long-term goal of understanding the mechanistic basis of miRNAs during development. The specific aims of the proposed research are designed to assess the regulatory roles of miRNAs during early embryonic differentiation events and, more generally, to test whether miRNAs initiate, maintain and/or fine-tune cell differentiation programs. By testing these hypotheses, insights into the functions of miRNAs in basic developmental processes will be generated. Cell-specific markers will first be examined in embryos with defects in miRNA biogenesis to identify the earliest stages and differentiation events that miRNAs are involved in. The corresponding miRNAs required for these cell-fate decisions will be identified using laser capture microdissection and highthroughput sequencing technologies. The localization patterns and functional domains of these early embryonic miRNAs will be characterized using RNA in situ hybridizations and miRNA sensor constructs. Candidate targets of early embryonic miRNAs will then be identified by determining which target transcripts are mis-regulated in miRNA biosynthesis mutants. The in vivo functions of miRNA/target interactions will be assessed using molecular genetic approaches. Characterizing the regulatory roles of miRNAs during cellular differentiation will contribute to a better understanding of miRNA functions. Since miRNA mis-regulation has been implicated in human disease, the proposed research may therefore yield insight into the mechanistic basis of disease-afflicted cell types.